Inhibitors of proteasomal activity for stimulating bone and hair growth

ABSTRACT

Compounds that inhibit the activity of NF-κB or inhibit the activity of the proteasome or both promote bone formation and hair growth and are thus useful in treating osteoporosis, bone fracture or deficiency, primary or secondary hyperparathyroidism, periodontal disease or defect, metastatic bone disease, osteolytic bone disease, post-plastic surgery, post-prosthetic joint surgery, and post-dental implantation; they also stimulate the production of hair follicles and are thus useful in stimulating hair growth, including hair density, in subject where this is desirable.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.09/421,545, filed Oct. 20, 1999, now pending, which is acontinuation-in-part of U.S. Ser. No. 09/361,775, filed Jul. 27, 1999,now pending, which is a continuation-in-part of U.S. Ser. No.09/113,947, filed Jul. 10, 1998, now pending. The contents of theseapplications are incorporated herein by reference.

TECHNICAL FIELD

[0002] The invention relates to compositions and methods for use intreating skeletal system disorders in a vertebrate at risk for boneloss, and in treating conditions that are characterized by the need forbone growth, in treating fractures, and in treating cartilage disorders.The invention also relates to enhancing hair density and growth. Morespecifically, the invention concerns the use of inhibitors ofproteasomal activity, e.g., inhibitors of the chymotrypsin-likeactivity, and inhibitors of NF-κB activity for enhancing hair growth.

BACKGROUND ART

[0003] Inhibitors of proteasomal activity, and to some extent inhibitorsof NF-κB activity, have two important physiological effects. First,proteasome inhibitors are able to enhance bone formation and are thususeful for treating various bone disorders. Second, both of theseinhibitors stimulate the production of hair follicles and are thususeful in stimulating hair growth, including hair density, in subjectwhere this is desirable.

[0004] Effect on Bone

[0005] Bone is subject to constant breakdown and resynthesis in acomplex process mediated by osteoblasts, which produce new bone, andosteoclasts, which destroy bone. The activities of these cells areregulated by a large number of cytokines and growth factors, many ofwhich have now been identified and cloned.

[0006] There is a plethora of conditions which are characterized by theneed to enhance bone formation or to inhibit bone resorption. Perhapsthe most obvious is the case of bone fractures, where it would bedesirable to stimulate bone growth and to hasten and complete bonerepair. Agents that enhance bone formation would also be useful infacial reconstruction procedures. Other bone deficit conditions includebone segmental defects, periodontal disease, metastatic bone disease,osteolytic bone disease and conditions where connective tissue repairwould be beneficial, such as healing or regeneration of cartilagedefects or injury. Also of great significance is the chronic conditionof osteoporosis, including age-related osteoporosis and osteoporosisassociated with post-menopausal hormone status. Other conditionscharacterized by the need for bone growth include primary and secondaryhyperparathyroidism, disuse osteoporosis, diabetes-related osteoporosis,and glucocorticoid-related osteoporosis.

[0007] There are currently no satisfactory pharmaceutical approaches tomanaging any of these conditions. Bone fractures are still treatedexclusively using casts, braces, anchoring devices and other strictlymechanical means. Further bone deterioration associated withpost-menopausal osteoporosis has been treated with estrogens orbisphosphonates, which may have drawbacks for some individuals. Althoughvarious approaches have been tried, as further discussed below, thereremains a need for additions to the repertoire of agents which can beused to treat these conditions.

[0008] Treatment of bone or other skeletal disorders, such as thoseassociated with cartilage, can be achieved either by enhancing boneformation or inhibiting bone resorption or both. A number of approacheshave been suggested which relate to bone formation.

[0009] Bone tissue is an excellent source for factors which have thecapacity for stimulating bone cells. Thus, extracts of bovine bonetissue obtained from slaughterhouses contain not only structuralproteins which are responsible for maintaining the structural integrityof bone, but also biologically active bone growth factors which canstimulate bone cells to proliferate. Among these latter factors aretransforming growth factor β, the heparin-binding growth factors (e.g.acidic and basic fibroblast growth factor), the insulin-like growthfactors (e.g., insulin-like growth factor I and insulin-like growthfactor II), and a recently described family of proteins called bonemorphogenetic proteins (BMPs). All of these growth factors have effectson other types of cells, as well as on bone cells.

[0010] The BMPs are novel factors in the extended transforming growthfactor B superfamily. Recombinant BMP2 and BMP4 can induce new boneformation when they are injected locally into the subcutaneous tissuesof rats (Wozney, J., Molec Reprod Dev (1992) 32:160-67). These factorsare expressed by normal osteoblasts as they differentiate, and have beenshown to stimulate osteoblast differentiation and bone nodule formationin vitro as well as bone formation in vivo (Harris S., et al., J BoneMiner Res (1994) 9:855-63). This latter property suggests potentialusefulness as therapeutic agents in diseases which result in bone loss.

[0011] The cells which are responsible for forming bone are osteoblasts.As osteoblasts differentiate from precursors to mature bone-formingcells, they express and secrete a number of enzymes and structuralproteins of the bone matrix, including Type-I collagen, osteocalcin,osteopontin and alkaline phosphatase. They also synthesize a number ofgrowth regulatory peptides which are stored in the bone matrix, and arepresumably responsible for normal bone formation. These growthregulatory peptides include the BMPs (Harris S., et al. (1994), supra).In studies of primary cultures of fetal rat calvarial osteoblasts, BMPs1, 2, 3, 4, and 6 are expressed by cultured cells prior to the formationof mineralized bone nodules (Harris S., et al (1994), supra). Likealkaline phosphatase, osteocalcin and osteopontin, the BMPs areexpressed by cultured osteoblasts as they proliferate and differentiate.

[0012] Although the BMPs are potent stimulators of bone formation invitro and in vivo, there are disadvantages to their use as therapeuticagents to enhance bone healing. Receptors for the bone morphogeneticproteins have been identified in many tissues, and the BMPs themselvesare expressed in a large variety of tissues in specific temporal andspatial patterns. This suggests that BMPs may have effects on manytissues in addition to bone, potentially limiting their usefulness astherapeutic agents when administered systemically. Moreover, since theyare peptides, they would have to be administered by injection. Thesedisadvantages impose severe limitations to the development of BMPs astherapeutic agents.

[0013] The fluorides, suggested also for this purpose, have a mode ofaction which may be related to tyrosine phosphorylation of growth factorreceptors on osteoblasts, as described, for example, Burgener, et al., JBone Min Res (1995) 10:164-171, but administration of fluorides isassociated with increased bone fragility, presumably due to effects onbone mineralization.

[0014] Small molecules which are able to stimulate bone formation havebeen disclosed in PCT applications WO98/17267 published Apr. 30, 1998,WO97/15308 published May 1, 1997 and WO97/48694 published Dec. 24, 1997.These agents generally comprise two aromatic systems spatially separatedby a linker. In addition, PCT application WO98/25460 published Jun. 18,1998 discloses the use of the class of compounds known as statins inenhancing bone formation. U.S. application Ser. No. 09/096,631 filedJun. 12, 1998 is directed to compounds for stimulating bone growth thatare generally isoprenoid pathway inhibitors. The contents of thisapplication, as well as that of the PCT applications cited above, areincorporated herein by reference.

[0015] Other agents appear to operate by preventing the resorption ofbone. Thus, U.S. Pat. No. 5,280,040 discloses compounds described asuseful in the treatment of osteoporosis. These compounds putativelyachieve this result by preventing bone resorption.

[0016] Wang, G. -J., et al., J Formos Med Assoc (1995) 94:589-592 reportthat certain lipid clearing agents, exemplified by lovastatin andbezafibrate, were able to inhibit the bone resorption resulting fromsteroid administration in rabbits. There was no effect on bone formationby these two compounds in the absence of steroid treatment. Themechanism of the inhibition in bone resorption observed in the presenceof steroids (and the mechanism of the effect of steroid on bone, per se)is said to be unknown.

[0017] An abstract entitled “Lovastatin Prevents Steroid-InducedAdipogenesis and Osteoporosis” by Cui, Q., et al, appeared in theReports of the ASBMR 18th Annual Meeting (September 1996) J Bone MineralRes. (1996)11 (S1):S510 which reports that lovastatin diminishedtriglyceride vesicles that accumulated when osteoprogenitor cells clonedfrom bone marrow stroma of chickens were treated in culture withdexamethasone. Lovastatin was reported to diminish the expression ofcertain mRNAs and to allow the cells to maintain the osteogenicphenotype after dexamethasone treatment, and chickens that had undergonebone loss in the femoral head as a result of dexamethasone treatmentwere improved by treatment with lovastatin.

[0018] These data are, however, contrary to reports that dexamethasoneand other inducers, such as BMPs, induce osteoblastic differentiationand stimulate osteocalcin mRNA (Bellows, C. G., et al., Develop Biol(1990) 140:132-38; Rickard, D. J., et al., Develop Biol (1994)161:218-28). In addition, Ducy, P., et al., Nature (1996) 382:448-52have recently reported that osteocalcin deficient mice exhibit aphenotype marked by increased bone formation and bones of improvedfunctional quality, without impairment of bone resorption. Ducy, et al.,state that their data suggest that osteocalcin antagonists may be oftherapeutic use in conjunction with estrogen replacement therapy (forprevention or treatment of osteoporosis).

[0019] It has also been shown that lovastatin inhibitslipopolysaccharide-induced NF-κB activation in human mesangial cells.Guijaro, C., et al, Nephrol Dial Transplant (1996) 11:6:990-996.

[0020] It has recently been shown that mice lacking expression of thetranscription factor NF-κB develop an abnormal bone condition,osteopetrosis (the converse of osteoporosis), due to an absence ofosteoclast formation (Franzoso, G., et al., Genes and Dev (1997)11:3482-3496; Iotsova, V., et al., Nature Med (1997) 3:1285-1289).Osteopetrosis is characterized by such an absence of osteoclast functionand the filling in of the marrow cavity with osteocartilagenousmaterial. The mice showed no abnormal osteoblast function. The abilityof proteasome inhibitors to stimulate bone growth is unexpected in lightof these results, where no effect on osteoblasts was shown sinceproteasome inhibitors are expected to function as NF-κB inhibitors aswell. This is because NF-κB must enter the nucleus to exert its effectson specific target genes, and compounds that inhibit its entry into thenucleus effectively inhibit its activity. Proteasome activity isrequired for NF-κB translocation. NF-κB is present in the cytoplasmbound to the inhibitory proteins IκBα and IκBβ which prevent itstranslocation. Translocation occurs when kinases phosphorylate IκBβ tocause its degradation by proteasome activity, thus resulting in itsrelease for entry into the nucleus. Inhibition of proteasome activityprevents this release and thus effectively inhibits NF-κB.

[0021] Effect on Hair Growth

[0022] Disorders of human hair growth include male pattern baldness,alopecia areota, alopecia induced by cancer chemotherapy and hairthinning associated with aging. These conditions are poorly understood,but nevertheless common and distressing, since hair is an importantfactor in human social and sexual communication.

[0023] Hair follicle regulation and growth are still not wellunderstood, but represent dynamic processes involving proliferation,differentiation and cellular interactions during tissue morphogenesis.It is believed that hair follicles are formed only in early stages ofdevelopment and not replaced.

[0024] Hardy, M. H., et al., Trans Genet (1992) 8:55-61 describesevidence that bone morphogenetic proteins (BMPs), members of the TGFβ,superfamily, are differentially expressed in hair follicles duringdevelopment. Harris, S. E., et al., J Bone Miner Res (1994) 9:855-863describes the effects of TGFβ on expression of BMP-2 and othersubstances in bone cells. BMP-2 expression in mature follicles alsooccurs during maturation and after the period of cell proliferation(Hardy, et al. (1992, supra). As noted, however, by Blessing, M., etal., Genes and Develop (1992) 7:204-215, the precise role functionalrole of BMP-2 in hair follicle maturation remains unclear.

[0025] Approaches to treat baldness abound in the U.S. patentliterature. See for example U.S. Pat. No. 5,767,152 (cyanocarboxylicacid derivatives), U.S. Pat. No. 5,824,643 (keratinocyte growth factors)and U.S. Pat. No. 5,910,497 (16-pyrazinyl-substitute-4-aza-androstane5-alpha.-reductase isozyme 1 inhibitors). There are many others.

[0026] Gat, U., et al., Cell (1998) 95:605-614 has demonstrated thatβ-catenin causes adult epithelial cells to create hair follicles, asurprising result in light of the known inability of mature cells to doso. B-Catenin is known to play a role in cell-cell adhesion and growthfactor signal transfection. It is also known that after ubiquitination,β-catenin is degraded by the proteasomes. Orford, K., et al., J BiolChem (1997) 272:24735-24738. At least one gene associated with hairgrowth (or lack thereof) has also been reported. Ahmed, W., et al,Science (1998) 279:720-724.

[0027] Two accepted agents currently used for the treatment of hair lossare the antihypertensive drug Minoxidil and the 5α-reductase inhibitorFinasteride. Neither is entirely satisfactory. Both suffer from modestefficacy and are inconvenient to administer. A specific, topicallyactive and easy to administer compound with better efficacy than theseagents would represent a marked advance.

[0028] Proteasomes and NF-κB

[0029] The present invention discloses convenient assays for compoundsthat will be useful in the treatment of bone disorders and instimulating hair growth. The assays involve inhibition of the activityof the transcription factor NF-κB or of the activity of proteasomalproteases, preferably proteasomal proteases. Compounds which inhibitthese activities are generally useful in treating hair growth disorders;proteasome inhibitors enhance bone growth. Compounds that inhibit theproduction of the transcription factor and these proteases will also beuseful in the invention. Their ability to do so can be further confirmedby additional assays.

[0030] The proteasome is a noncompartmentalized collection of unrelatedproteases which form a common architecture in which proteolytic subunitsare self-assembled to form barrel-shaped complexes (for review, seeBaumeister, et al., Cell (1998) 92:367-380. The proteasome contains anarray of distinct proteolytic activities inside eucaryotic cells.Compounds which inhibit proteasomal activity also reduce NF-κB activityby limiting its capacity to be translocated to the nucleus (Barnes, P.J., et al., New Engl J Med (1997) 336:1066-1071.

DISCLOSURE OF THE INVENTION

[0031] The present invention adds to the repertoire of osteogenic andhair growth stimulating agents by providing drugs which would inhibitkey proteins and enzymes involved in proteasomal activity and whichdecrease the activity of the nuclear transcription factor NF-κB, andthus stimulate bone or hair growth. In accordance with the presentinvention, we have discovered that inhibition of the functions of theproteasomal proteins and, to a lesser extent, of NF-κB in bone cellsleads to increased bone growth and to hair follicle formation andstimulation; the effect on hair is also exhibited by inhibitors ofNF-κB. Thus, assessing a candidate compound for its ability to inhibitproteasomal proteins or NF-κB provides a useful means to identify boneand hair growth anabolic agents.

[0032] The present specification thus provides methods foridentification of osteogenic compounds to stimulate bone growth andcompounds that stimulate hair growth by assessing their capacity toinhibit proteasome activity and to stimulate hair growth by assessingtheir ability to inhibit the activity of the transcription factor NF-κB,preferably to inhibit proteasomal activity. Also useful in the methodsof the invention are compounds which inhibit the in situ production ofthe enzymes contained in the proteasome or inhibit the production ofNF-κB, preferably of enzymes of the proteasomes. Once a compound foundto inhibit these activities has been identified, it can be used in anadditional aspect of the invention—a method to stimulate the growth ofbone or of hair by contacting suitable cells with the identifiedcompound. The cellular contact may include in vivo administration andthe compounds of the invention are thus useful in treating degenerativebone diseases, fractures, dental problems, baldness, alopecia and thelike. These methods are performed, according to the present invention,with compounds identified as inhibitors of proteasome activity orinhibitors of the activity of transcription factor NF-κB, preferablyinhibitors of the proteasome enzymes, or inhibitors of the production ofthe proteasome enzymes or of NF-κB, preferably of the proteasomeenzymes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 shows a diagram of the isoprenoid pathway.

MODES OF CARRYING OUT THE INVENTION

[0034] In accordance with the present invention, there are providedmethods of treating bone defects (including osteoporosis, fractures,osteolytic lesions and segmental bone defects) in subjects sufferingtherefrom said method comprising administering to said subject, in anamount sufficient to stimulate bone growth, a compound which inhibitsproteasomal activity and function or the production of this protein.Inhibitors of NF-κB are also implicated.

[0035] Also in accordance with the present invention, there are providedmethods of treating disorders of hair growth. Disorders of hair growthmay be the result of a defect in the ability of existing hair folliclesto extrude hair, or may be the result of a deficiency in the number ofhair follicles per se. “Stimulation of hair growth” refers to increasingthe volume of hair in a particular area of a subject whether this is theresult of an increased rate of growth in length and/or thickness fromthe same number of hair follicles, growth proceeding from an enhancednumber of hair follicles, or both. The number of hair follicles can beenhanced by further activating existing hair follicles or by stimulatingthe appearance or proliferation of hair follicles in a particular regionof the skin.

[0036] As employed herein, the term “subject” embraces human as well asother animal species, such as, for example, canine, feline, bovine,porcine, rodent, and the like. It will be understood by the skilledpractitioner that the subject is one appropriate to the desirability ofstimulating bone growth or hair growth. Thus, in general, for example,stimulation of hair growth will be confined in most instances to animalsthat would appropriately exhibit such growth.

[0037] As used herein, “treat” or “treatment” include a postponement ofdevelopment of bone deficit symptoms and/or a reduction in the severityof such symptoms that will or are expected to develop. These termsfurther include ameliorating existing bone or cartilage deficitsymptoms, preventing additional symptoms, ameliorating or preventing theunderlying metabolic causes of symptoms, preventing or reversing boneresorption and/or encouraging bone growth. Thus, the terms denote that abeneficial result has been conferred on a vertebrate subject with acartilage, bone or skeletal deficit, or with the potential to developsuch deficit.

[0038] By “bone deficit” is meant an imbalance in the ratio of boneformation to bone resorption, such that, if unmodified, the subject willexhibit less bone than desirable, or the subject's bones will be lessintact and coherent than desired. Bone deficit may also result fromfracture, from surgical intervention or from dental or periodontaldisease. By “cartilage defect” is meant damaged cartilage, lesscartilage than desired, or cartilage that is less intact and coherentthan desired. “Bone disorders” includes both bone deficits and cartilagedefects.

[0039] Representative uses of the compounds identified by the assay ofthe invention include: repair of bone defects and deficiencies, such asthose occurring in closed, open and non-union fractures; prophylacticuse in closed and open fracture reduction; promotion of bone healing inplastic surgery; stimulation of bone in-growth into non-cementedprosthetic joints and dental implants; elevation of peak bone mass inpre-menopausal women; treatment of growth deficiencies; treatment ofperiodontal disease and defects, and other tooth repair processes;increase in bone formation during distraction osteogenesis; andtreatment of other skeletal disorders, such as age-related osteoporosis,post-menopausal osteoporosis, glucocorticoid-induced osteoporosis ordisuse osteoporosis and arthritis, or any condition that benefits fromstimulation of bone formation. The compounds of the present inventioncan also be useful in repair of congenital, trauma-induced or surgicalresection of bone (for instance, for cancer treatment), and in cosmeticsurgery. Further, the compounds of the present invention can be used forlimiting or treating cartilage defects or disorders, and may be usefulin wound healing or tissue repair.

[0040] Conditions which would be benefited by “treating” or “treatment”for stimulation of hair growth include male pattern baldness, alopeciacaused by chemotherapy, hair thinning resulting from aging, geneticdisorders which result in deficiency of hair coverage, and, in animals,providing additional protection from cold temperatures. Thus, while usein humans may be primarily of cosmetic benefit, use in animals may betherapeutic as well.

[0041] The compositions of the invention may be administeredsystemically or locally. For systemic use, the compounds herein areformulated for parenteral (e.g., intravenous, subcutaneous,intramuscular, intraperitoneal, intranasal or transdermal) or enteral(e.g., oral or rectal) delivery according to conventional methods.Intravenous administration can be by a series of injections or bycontinuous infusion over an extended period. Administration by injectionor other routes of discretely spaced administration can be performed atintervals ranging from weekly to once to three times daily.Alternatively, the compounds disclosed herein may be administered in acyclical manner (administration of disclosed compound; followed by noadministration; followed by administration of disclosed compound, andthe like). Treatment will continue until the desired outcome isachieved. In general, pharmaceutical formulations will include acompound of the present invention in combination with a pharmaceuticallyacceptable vehicle, such as saline, buffered saline, 5% dextrose inwater, borate-buffered saline containing trace metals or the like.Formulations may further include one or more excipients, preservatives,solubilizers, buffering agents, albumin to prevent protein loss on vialsurfaces, lubricants, fillers, stabilizers, etc. Methods of formulationare well known in the art and are disclosed, for example, in Remington'sPharmaceutical Sciences, latest edition, Mack Publishing Co., EastonPa., which is incorporated herein by reference. Pharmaceuticalcompositions for use within the present invention can be in the form ofsterile, non-pyrogenic liquid solutions or suspensions, coated capsules,suppositories, lyophilized powders, transdermal patches or other formsknown in the art. Local administration may be by injection at the siteof injury or defect, or by insertion or attachment of a solid carrier atthe site, or by direct, topical application of a viscous liquid, or thelike. For local administration, the delivery vehicle preferably providesa matrix for the growing bone or cartilage, and more preferably is avehicle that can be absorbed by the subject without adverse effects.

[0042] Delivery of compounds herein to wound sites may be enhanced bythe use of controlled-release compositions, such as those described inPCT publication WO93/20859, which is incorporated herein by reference.Films of this type are particularly useful as coatings for prostheticdevices and surgical implants. The films may, for example, be wrappedaround the outer surfaces of surgical screws, rods, pins, plates and thelike. Implantable devices of this type are routinely used in orthopedicsurgery. The films can also be used to coat bone filling materials, suchas hydroxyapatite blocks, demineralized bone matrix plugs, collagenmatrices and the like. In general, a film or device as described hereinis applied to the bone at the fracture site. Application is generally byimplantation into the bone or attachment to the surface using standardsurgical procedures.

[0043] In addition to the copolymers and carriers noted above, thebiodegradable films and matrices may include other active or inertcomponents. Of particular interest are those agents that promote tissuegrowth or infiltration, such as growth factors. Exemplary growth factorsfor this purpose include epidermal growth factor (EGF), fibroblastgrowth factor (FGF), platelet-derived growth factor (PDGF), transforminggrowth factors (TGFs), parathyroid hormone (PTH), leukemia inhibitoryfactor (LIF), insulin-like growth factors (IGFs) and the like. Agentsthat promote bone growth, such as bone morphogenetic proteins (U.S. Pat.No. 4,761,471; PCT Publication WO90/11366), osteogenin (Sampath, et al.,Proc. Natl. Acad. Sci. USA (1987) 84:7109-13) and NaF (Tencer, et al., JBiomed. Mat. Res. (1989) 23: 571-89) are also preferred. Biodegradablefilms or matrices include calcium sulfate, tricalcium phosphate,hydroxyapatite, polylactic acid, polyanhydrides, bone or dermalcollagen, pure proteins, extracellular matrix components and the likeand combinations thereof Such biodegradable materials may be used incombination with non-biodegradable materials, to provide desiredmechanical, cosmetic or tissue or matrix interface properties.

[0044] Alternative methods for delivery of compounds of the presentinvention include use of ALZET osmotic minipumps (Alza Corp., Palo Alto,Calif.); sustained release matrix materials such as those disclosed inWang, et al. (PCT Publication WO90/11366); electrically charged dextranbeads, as disclosed in Bao, et al. (PCT Publication WO92/03125);collagen-based delivery systems, for example, as disclosed in Ksander,et al, Ann. Surg. (1990) 211(3):288-94; methylcellulose gel systems, asdisclosed in Beck, et al., J Bone Min. Res. (1991) 6(11):1257-65;alginate-based systems, as disclosed in Edelman, et al., Biomaterials(1991) 12:619-26 and the like. Other methods well known in the art forsustained local delivery in bone include porous coated metal prosthesesthat can be impregnated and solid plastic rods with therapeuticcompositions incorporated within them.

[0045] The compounds of the present invention may also be used inconjunction with agents that inhibit bone resorption. Antiresorptiveagents, such as estrogen, bisphosphonates and calcitonin, are preferredfor this purpose. More specifically, the compounds disclosed herein maybe administered for a period of time (for instance, months to years)sufficient to obtain correction of a bone deficit condition. Once thebone deficit condition has been corrected, the vertebrate can beadministered an anti-resorptive compound to maintain the corrected bonecondition. Alternatively, the compounds disclosed herein may beadministered with an anti-resorptive compound in a cyclical manner(administration of disclosed compound, followed by anti-resorptive,followed by disclosed compound, and the like).

[0046] In additional formulations, conventional preparations such asthose described below may be used.

[0047] Aqueous suspensions may contain the active ingredient inadmixture with pharmacologically acceptable excipients, comprisingsuspending agents, such as methyl cellulose; and wetting agents, such aslecithin, lysolecithin or long-chain fatty alcohols. The said aqueoussuspensions may also contain preservatives, coloring agents, flavoringagents, sweetening agents and the like in accordance with industrystandards.

[0048] Preparations for topical and local application comprise aerosolsprays, lotions, gels and ointments in pharmaceutically appropriatevehicles which may comprise lower aliphatic alcohols, polyglycols suchas glycerol, polyethylene glycol, esters of fatty acids, oils and fats,and silicones. The preparations may further comprise antioxidants, suchas ascorbic acid or tocopherol, and preservatives, such asp-hydroxybenzoic acid esters.

[0049] Parenteral preparations comprise particularly sterile orsterilized products. Injectable compositions may be provided containingthe active compound and any of the well known injectable carriers. Thesemay contain salts for regulating the osmotic pressure.

[0050] If desired, the osteogenic agents can be incorporated intoliposomes by any of the reported methods of preparing liposomes for usein treating various pathogenic conditions. The present compositions mayutilize the compounds noted above incorporated in liposomes in order todirect these compounds to macrophages, monocytes, as well as other cellsand tissues and organs which take up the liposomal composition. Theliposome-incorporated compounds of the invention can be utilized byparenteral administration, to allow for the efficacious use of lowerdoses of the compounds. Ligands may also be incorporated to furtherfocus the specificity of the liposomes.

[0051] Suitable conventional methods of liposome preparation include,but are not limited to, those disclosed by Bangham, A. D., et al., J MolBiol (1965) 23:238-252, Olson, F., et al., Biochim Biophys Acta (1979)557:9-23, Szoka, F., et al., Proc Natl Acad Sci USA (1978)75:4194-4198,Kim, S., et al., Biochim Biophys Acta (1983) 728:339:348, and Mayer, etal., Biochim Biophys Acta (1986)858:161-168.

[0052] The liposomes may be made from the present compounds incombination with any of the conventional synthetic or naturalphospholipid liposome materials including phospholipids from naturalsources such as egg, plant or animal sources such asphosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol,sphingomyelin, phosphatidylserine, or phosphatidylinositol and the like.Synthetic phospholipids that may also be used, include, but are notlimited to: dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidycholine, and thecorresponding synthetic phosphatidylethanolamines andphosphatidylglycerols. Cholesterol or other sterols, cholesterolhemisuccinate, glycolipids, cerebrosides, fatty acids, gangliosides,sphingolipids, 1,2-bis(oleoyloxy)-3-(trimethyl ammonio) propane (DOTAP),N-[1-(2,3-dioleoyl) propyl-N,N,N-trimethylammonium chloride (DOTMA), andother cationic lipids may be incorporated into the liposomes, as isknown to those skilled in the art. The relative amounts of phospholipidand additives used in the liposomes may be varied if desired. Thepreferred ranges are from about 60 to 90 mole percent of thephospholipid; cholesterol, cholesterol hemisuccinate, fatty acids orcationic lipids may be used in amounts ranging from 0 to 50 molepercent. The amounts of the present compounds incorporated into thelipid layer of liposomes can be varied with the concentration of thelipids ranging from about 0.01 to about 50 mole percent.

[0053] The liposomes with the above formulations may be made still morespecific for their intended targets with the incorporation of monoclonalantibodies or other ligands specific for a target. For example,monoclonal antibodies to the BMP receptor may be incorporated into theliposome by linkage to phosphatidylethanolamine (PE) incorporated intothe liposome by the method of Leserman, L., et al., Nature (1980)288:602-604.

[0054] Veterinary uses of the disclosed compounds are also contemplated,as set forth above. Such uses would include treatment of bone orcartilage deficits or defects associated with hair or fur in domesticanimals, livestock and thoroughbred horses.

[0055] The compounds of the present invention may be used to stimulategrowth of bone-forming cells or their precursors, or to inducedifferentiation of bone-forming cell precursors, either in vitro or exvivo. The compounds described herein may also modify a target tissue ororgan environment, so as to attract bone-forming cells to an environmentin need of such cells. As used herein, the term “precursor cell” refersto a cell that is committed to a differentiation pathway, but thatgenerally does not express markers or function as a mature, fullydifferentiated cell. As used herein, the term “mesenchymal cells” or“mesenchymal stem cells” refers to pluripotent progenitor cells that arecapable of dividing many times, and whose progeny will give rise toskeletal tissues, including cartilage, bone, tendon, ligament, marrowstroma and connective tissue (see A. Caplan, J Orthop. Res. (1991)9:641-50). As used herein, the term “osteogenic cells” includesosteoblasts and osteoblast precursor cells. More particularly, thedisclosed compounds are useful for stimulating a cell populationcontaining marrow mesenchymal cells, thereby increasing the number ofosteogenic cells in that cell population. In a preferred method,hematopoietic cells are removed from the cell population, either beforeor after stimulation with the disclosed compounds. Through practice ofsuch methods, osteogenic cells may be expanded. The expanded osteogeniccells can be infused (or reinfused) into a vertebrate subject in needthereof. For instance, a subject's own mesenchymal stem cells can beexposed to compounds of the present invention ex vivo, and the resultantosteogenic cells could be infused or directed to a desired site withinthe subject, where further proliferation and/or differentiation of theosteogenic cells can occur without immunorejection. Alternatively, thecell population exposed to the disclosed compounds may be immortalizedhuman fetal osteoblastic or osteogenic cells. If such cells are infusedor implanted in a vertebrate subject, it may be advantageous to“immunoprotect” these non-self cells, or to immunosuppress (preferablylocally) the recipient to enhance transplantation and bone or cartilagerepair.

[0056] As stated above, the compounds of the present invention may alsobe used to stimulate the growth of hair either by enhancing its rate offormation from existing follicles, stimulating inactive follicles,effecting the production of additional hair follicles or somecombination of the foregoing, or by any other mechanism that may or maynot presently be understood.

[0057] Within the present invention, an “effective amount” of acomposition is that amount which produces a statistically significanteffect. For example, an “effective amount” for therapeutic uses is theamount of the composition comprising an active compound herein requiredto provide a clinically significant increase in healing rates infracture repair; reversal of bone loss in osteoporosis; reversal ofcartilage defects or disorders; prevention or delay of onset ofosteoporosis; stimulation and/or augmentation of bone formation infracture non-unions and distraction osteogenesis; increase and/oracceleration of bone growth into prosthetic devices; and repair ofdental defects. An “effective amount” for uses in stimulating hairgrowth is that amount which provides the desired effect in terms oflength or density of hair. Such effective amounts will be determinedusing routine optimization techniques and are dependent on theparticular condition to be treated, the condition of the patient, theroute of administration, the formulation, and the judgment of thepractitioner and other factors evident to those skilled in the art. Thedosage required for the compounds of the invention (for example, inosteoporosis where an increase in bone formation is desired) ismanifested as a statistically significant difference in bone massbetween treatment and control groups. This difference in bone mass maybe seen, for example, as a 5-20% or more increase in bone mass in thetreatment group. Other measurements of clinically significant increasesin healing may include, for example, tests for breaking strength andtension, breaking strength and torsion, 4-point bending, increasedconnectivity in bone biopsies and other biomechanical tests well knownto those skilled in the art. General guidance for treatment regimens isobtained from experiments carried out in animal models of the disease ofinterest. Differences between successfully treated subjects and controlswith regard to stimulation of hair growth can generally be ascertainedby direct observation.

[0058] The dosage of the compounds of the invention will vary accordingto the extent and severity of the need for treatment, the activity ofthe administered compound, the general health of the subject, and otherconsiderations well known to the skilled artisan. Generally, they can beadministered to a typical human on a daily basis as an oral dose ofabout 0.1 mg/kg-1000 mg/kg, and more preferably from about 1 mg/kg toabout 200 mg/kg. The parenteral dose will appropriately be 20-100% ofthe oral dose. While oral administration may be preferable in mostinstances where the condition is a bone deficit (for reasons of ease,patient acceptability, and the like), alternative methods ofadministration may be appropriate for selected compounds and selecteddefects or diseases. While topical administration is generallypreferable for stimulating hair growth, as generally only local effectsare desired, systemic treatment may be preferable in some instances aswell.

[0059] Assays for Compounds Useful in the Invention

[0060] Assays for assessing the ability of a compound to inhibitproteasomal activity and for inhibitors of NF-κB activity are well knownin the art. Two typical, but nonlimiting assays are described below.

[0061] Assessment of Proteasomal Activity

[0062] Proteasomal inhibition activity is most conveniently measured bythe assay described in Example 5 hereinbelow. The assay involvesincubating the potential inhibitor with 20S thermophila proteasomeswhich, in purified form, are commercially available, with a fluorogenicpeptide substrate. The presence of an inhibitor will reduce the amountof fluorescence generated by the action of the proteasome fraction onthe fluorogenic peptide. This assay is described in further detail inCoux, O., et al., N Rev Biochem (1996) 65:801; Adams, J., et al., CancerRes (1999) 59:2615; and Craiu, A., et al., J Biol Chem (1997) 272:13437.Further reports are set forth in Hilt, W., et al., Trans Biochem Sci(1996) 21:96; Peters, J., Trends Biochem Sci (1994) 19:377;Maupin-Furlow, J. A., et al., J Biol Chem (1995) 270:28617; and Jensen,T. J., et al., Cell (1995) 83:129. Fluorogenic substrates and purifiedproteasomes are available, for example, from CalBiochem, San Diego,Calif.

[0063] NF-κB Activity Assays

[0064] Cells are treated with different concentrations of compounds, andnuclear extracts prepared. Briefly, cells are washed withphosphate-buffered saline, and resuspended in lysis buffer (0.6% NonidetP-40, 150 mM NaCl, 10 mM Tris-HCl, pH 7.9, 1 mM EDTA, 0.5 mM DTT and acocktail of protease inhibitors (Complete (TM), Boehringer Mannheim).After incubation on ice for 15 min, nuclei are collected bycentrifugation. The pellet is resuspended in nuclear extraction buffer(10 mM Hepes, pH 7.9, 420 mM NaCl, 0.1 mM EDTA, 1.5 mM NgCl₂, 0.5 mMDTT, protease inhibitors (Complete (TM), Boehringer Mannheim), 25%glycerol), and incubated at 4 degrees C. for 30 min. The supernatant iscollected and dialyzed in a buffer containing 10 mM Tris-HCl, pH 7.5, 50mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM DTT, and 20% glycerol. Afterdialysis, the nuclear extract is centrifuged to remove precipitatedproteins, and aliquots are stored at −70 C. Protein concentration in thenuclear extracts is measured by the method of Bradford using adye-binding assay kit (Bio-Rad).

[0065] The probe for electrophoretic mobility shift assays is a32P-labeled double-stranded oligonucleotide containing the consensussequence specific for NF-κB (Promega). Nuclear extracts (5 ug) arepre-incubated in 20-ul reaction mixtures containing 10 mM Tris-HCl, pH7.5, 50 mM NaCl, 2.5 mM DTT, 0.5 mM EDTA, 1 mM MgCl₂, 4% glycerol, and 5μg of poly (dI-dC). After 10 min at room temperature, 10-20 fmol ofprobe is added, and incubated further for 20 min. DNA-protein complexesare separated from free oligonucleotides on a 5% polyacrylamide/0.5× TBEgel (45 mM Tris-HCl, 45 mM boric acid, 1 mM EDTA). Afterelectrophoresis, gels are dried and autoradiographed.

[0066] Assays for Production Inhibition

[0067] Compounds which inhibit the production of the enzymes havingproteasomal activity or of NF-κB can be assessed by measuring the levelof production of these proteins in the presence and absence of candidatecompounds. The levels of production can be readily measured in in vitrosystems using, for example, immunoassays for the level of proteinproduced. The levels of such proteins can also be assessed by utilizing,for example, methionine labeling and size separation of proteins in thecells to be assessed. In order to effect a convenient level of proteinproduction for measurement, it is advantageous to use recombinantexpression systems for the relevant enzymes or the NF-κB so thatsubstantial amounts are produced.

[0068] Typical approaches to inhibiting the production of NF-κB orproteasome enzymes include the use of antisense technology or formationof triplexes with double-stranded forms of nucleotide sequences relevantin the expression of the genes. In addition, various small molecules mayalso inhibit this production.

[0069] Screening Assays—Bone

[0070] The osteogenic activity of the compounds used in the methods ofthe invention can be verified using in vitro screening techniques, suchas the assessment of transcription of a reporter gene coupled to a bonemorphogenetic protein-associated promoter or in alternative assays.

[0071] ABA Screening Assay

[0072] A rapid throughput screening test for compounds that stimulatebone formation by demonstration that they are capable of stimulatingexpression of a reporter gene linked to a BMP promoter (a surrogate forthe production of bone morphogenetic factors that are endogenouslyproduced) is described in U.S. application Ser. No. 08/458,434, filedJune 2, 1995, the entire contents of which are incorporated herein byreference. This assay is also described as a portion of a study ofimmortalized murine osteoblasts (derived from a mouse expressing atransgene composed of a BMP2 promoter driving expression of T-antigen)in Ghosh-Choudhery, N., et al, Endocrinology (1996) 137:331-39. In thisstudy, the immortalized cells were stably transfected with a plasmidcontaining a luciferase reporter gene driven by a mouse BMP2 promoter(−2736/114 bp), and responded in a dose-dependent manner to recombinanthuman BMP2.

[0073] Briefly, the assay utilizes cells transformed permanently ortransiently with constructs in which the promoter of a bonemorphogenetic protein, specifically BMP2 or BMP4, is coupled to areporter gene, typically luciferase. These transformed cells are thenevaluated for the production of the reporter gene product; compoundsthat activate the BMP promoter will drive production of the reporterprotein, which can be readily assayed. Many thousands of compounds havebeen subjected to this rapid screening technique, and only a very smallpercentage are able to elicit a level of expression of reporter gene5-fold greater than that produced by vehicle. Compounds that activatethe BMP promoter fall into groups, where members of each group sharecertain structural characteristics not present in inactive compounds.The active compounds (“BMP promoter-active compounds” or “activecompounds”) are useful in promoting bone or cartilage growth, and thusin the treatment of vertebrates in need of bone or cartilage growth.

[0074] BMP promoter-active compounds can be examined in a variety ofother assays that test specificity and toxicity. For instance, non-BMPpromoters or response elements can be linked to a reporter gene andinserted into an appropriate host cell. Cytotoxicity can be determinedby visual or microscopic examination of BMP promoter—and/or non-BMPpromoter-reporter gene-containing cells, for instance. Alternatively,nucleic acid and/or protein synthesis by the cells can be monitored. Forin vivo assays, tissues may be removed and examined visually ormicroscopically, and optionally examined in conjunction with dyes orstains that facilitate histologic examination. In assessing in vivoassay results, it may also be useful to examine biodistribution of thetest compound, using conventional medicinal chemistry/animal modeltechniques.

[0075] Neonatal Mouse Calvaria Assay (In vitro)

[0076] An assay for bone resorption or bone formation is similar to thatdescribed by Gowen M. & Mundy G., J. Immunol (1986) 136:2478-82.Briefly, four days after birth, the front and parietal bones of ICRSwiss white mouse pups are removed by microdissection and split alongthe sagittal suture. In an assay for resorption, the bones are incubatedin BGJb medium (Irvine Scientific, Santa Ana, Calif.) plus 0.02% (orlower concentration) β-methylcyclodextrin, wherein the medium alsocontains test or control substances. The medium used when the assay isconducted to assess bone formation is Fitton and Jackson Modified BGJMedium (Sigma) supplemented with 6 μg/ml insulin, 6 μg/ml transferrin, 6ng/ml selenous acid, calcium and phosphate concentrations of 1.25 and3.0 mM, respectively, and ascorbic acid to a concentration of 100 μg/mlis added every two days. The incubation is conducted at 37° C. in ahumidified atmosphere of 5% CO₂ and 95% air for 96 hours.

[0077] Following this, the bones are removed from the incubation mediaand fixed in 10% buffered formalin for 24-48 hours, decalcified in 14%EDTA for 1 week, processed through graded alcohols; and embedded inparaffin wax. Three μm sections of the calvaria are prepared.Representative sections are selected for histomorphometric assessment ofbone formation or bone resorption. Bone changes are measured on sectionscut 200 μm apart. Osteoblasts and osteoclasts are identified by theirdistinctive morphology.

[0078] BMP promoter-active compounds can be examined in a variety ofother assays that test specificity and toxicity. For instance, non-BMPpromoters or response elements can be linked to a reporter gene andinserted into an appropriate host cell. Cytotoxicity can be determinedby visual or microscopic examination of BMP promoter- and/or non-BMPpromoter-reporter gene-containing cells, for instance. Alternatively,nucleic acid and/or protein synthesis by the cells can be monitored. Forin vivo assays, tissues may be removed and examined visually ormicroscopically, and optionally examined in conjunction with dyes orstains that facilitate histologic examination. In assessing in vivoassay results, it may also be useful to examine biodistribution of thetest compound, using conventional medicinal chemistry/animal modeltechniques.

[0079] Neonatal Mouse Calvaria Assay (In vitro)

[0080] An assay for bone resorption or bone formation is similar to thatdescribed by Gowen M. & Mundy G., J Immunol (1986) 136:2478-82. Briefly,four days after birth, the front and parietal bones of ICR Swiss whitemouse pups are removed by microdissection and split along the sagittalsuture. In an assay for resorption, the bones are incubated in BGJbmedium (Irvine Scientific, Santa Ana, Calif.) plus 0.02% (or lowerconcentration) β-methylcyclodextrin, wherein the medium also containstest or control substances. The medium used when the assay is conductedto assess bone formation is Fitton and Jackson Modified BGJ Medium(Sigma) supplemented with 6 μg/ml insulin, 6 μg/ml transferrin, 6 μg/mlselenous acid, calcium and phosphate concentrations of 1.25 and 3.0 mM,respectively, and ascorbic acid to a concentration of 100 μg/ml is addedevery two days. The incubation is conducted at 37° C. in a humidifiedatmosphere of 5% CO₂ and 95% air for 96 hours.

[0081] Following this, the bones are removed from the incubation mediaand fixed in 10% buffered formalin for 24-48 hours, decalcified in 14%EDTA for 1 week, processed through graded alcohols; and embedded inparaffin wax. Three μm sections of the calvaria are prepared.Representative sections are selected for histomorphometric assessment ofbone formation or bone resorption. Bone changes are measured on sectionscut 200 μm apart. Osteoblasts and osteoclasts are identified by theirdistinctive morphology.

[0082] Other auxiliary assays can be used as controls to determinenon-BMP promoter-mediated effects of test compounds. For example,mitogenic activity can be measured using screening assays featuring aserum-response element (SRE) as a promoter and a luciferase reportergene. More specifically, these screening assays can detect signalingthrough SRE-mediated pathways, such as the protein kinase C pathway. Forinstance, an osteoblast activator SRE-luciferase screen and an insulinmimetic SRE-luciferase screen are useful for this purpose. Similarly,test compound stimulation of cAMP response element (CRE)-mediatedpathways can also be assayed. For instance, cells transfected withreceptors for PTH and calcitonin (two bone-active agents) can be used inCRE-luciferase screens to detect elevated cAMP levels. Thus, the BMPpromoter specificity of a test compound can be examined through use ofthese types of auxiliary assays.

[0083] In vivo Assay of Effects of Compounds on Murine Calvarial BoneGrowth

[0084] Male ICR Swiss white mice, aged 4-6 weeks and weighing 13-26 gm,are employed, using 4-5 mice per group. The calvarial bone growth assayis performed as described in PCT application WO95/24211, incorporated byreference. Briefly, the test compound or appropriate control vehicle isinjected into the subcutaneous tissue over the right calvaria of normalmice. Typically, the control vehicle is the vehicle in which thecompound was solubilized, and is PBS containing 5% DMSO or is PBScontaining Tween (2 μl/10 ml). The animals are sacrificed on day 14 andbone growth measured by histomorphometry. Bone samples for quantitationare cleaned from adjacent tissues and fixed in 10% buffered formalin for24-48 hours, decalcified in 14% EDTA for 1-3 weeks, processed throughgraded alcohols; and embedded in paraffin wax. Three to five μm sectionsof the calvaria are prepared, and representative sections are selectedfor histomorphometric assessment of the effects on bone formation andbone resorption. Sections are measured by using a camera lucidaattachment to trace directly the microscopic image onto a digitizingplate. Bone changes are measured on sections cut 200 μm apart, over 4adjacent 1×1 mm fields on both the injected and noninjected sides of thecalvaria. New bone is identified by its characteristic woven structure,and osteoclasts and osteoblasts are identified by their distinctivemorphology. Histomorphometry software (OsteoMeasure, Osteometrix, Inc.,Atlanta) is used to process digitizer input to determine cell counts andmeasure areas or perimeters.

[0085] Typical treatment regimens for testing utilize application of thecompound to be tested over several days of repeated administration.

[0086] Additional In Vivo Assa Bone

[0087] Lead compounds can be further tested in intact animals using anin vivo, dosing assay. Prototypical dosing may be accomplished bysubcutaneous, intraperitoneal or oral administration, and may beperformed by injection, sustained release or other delivery techniques.The time period for administration of test compound may vary (forinstance, 28 days as well as 35 days may be appropriate). An exemplary,in vivo oral or subcutaneous dosing assay may be conducted as follows:

[0088] In a typical study, 70 three-month-old female Sprague-Dawley ratsare weight-matched and divided into seven groups, with ten animals ineach group. This includes a baseline control group of animals sacrificedat the initiation of the study; a control group administered vehicleonly; a PBS-treated control group; and a positive control groupadministered a compound (non-protein or protein) known to promote bonegrowth. Three dosage levels of the compound to be tested areadministered to the remaining three groups.

[0089] Briefly, test compound, positive control compound, PBS, orvehicle alone is administered subcutaneously once per day for 35 days.All animals are injected with calcein nine days and two days beforesacrifice (two injections of calcein administered each designated day).Weekly body weights are determined. At the end of the 35-day cycle, theanimals are weighed and bled by orbital or cardiac puncture. Serumcalcium, phosphate, osteocalcin, and CBCs are determined. Both leg bones(femur and tibia) and lumbar vertebrae are removed, cleaned of adheringsoft tissue, and stored in 70% ethanol for evaluation, as performed byperipheral quantitative computed tomography (PQCT; Ferretti, J., Bone(1995) 17:353S-64S), dual energy X-ray absorptiometry (DEXA;Laval-Jeantet A., et al., Calcif Tissue Intl (1995) 56:14-18; J. Casez,et al, Bone and Mineral (1994) 26:61-68) and/or histomorphometry. Theeffect of test compounds on bone remodeling can thus be evaluated.

[0090] Lead compounds can also be tested in acute ovariectomized animals(prevention model) using an in vivo dosing assay. Such assays may alsoinclude an estrogen-treated group as a control. An exemplarysubcutaneous dosing assay is performed as follows:

[0091] In a typical study, 80 three-month-old female Sprague-Dawley ratsare weight-matched and divided into eight groups, with ten animals ineach group. This includes a baseline control group of animals sacrificedat the initiation of the study; three control groups (shamovariectomized (sham OVX)+vehicle only; ovariectomized (OVX)+vehicleonly; PBS-treated OVX); and a control OVX group that is administered acompound known to promote bone growth. Three dosage levels of thecompound to be tested are administered to the remaining three groups ofOVX animals.

[0092] Since ovariectomy (OVX) induces hyperphagia, all OVX animals arepair-fed with sham OVX animals throughout the 35 day study. Briefly,test compound, positive control compound, PBS, or vehicle alone isadministered orally or subcutaneously once per day for 35 days.Alternatively, test compound can be formulated in implantable pelletsthat are implanted for 35 days, or may be administered orally, such asby gastric gavage. All animals, including sham OVX/vehicle andOVX/vehicle groups, are injected intraperitoneally with calcein ninedays and two days before sacrifice (two injections of calceinadministered each designated day, to ensure proper labeling of newlyformed bone). Weekly body weights are determined. At the end of the35-day cycle, the animals' blood and tissues are processed as describedabove.

[0093] Lead compounds may also be tested in chronic OVX animals(treatment model).

[0094] An exemplary protocol for treatment of established bone loss inovariectomized animals that can be used to assess efficacy of anabolicagents may be performed as follows.

[0095] Briefly, 80 to 100 six month old female, Sprague-Dawley rats aresubjected to sham surgery (sham OVX) or ovariectomy (OVX) at time 0, and10 rats are sacrificed to serve as baseline controls. Body weights arerecorded weekly during the experiment. After approximately 6 weeks (42days) or more of bone depletion, 10 sham OVX and 10 OVX rats arerandomly selected for sacrifice as depletion period controls. Of theremaining animals, 10 sham OVX and 10 OVX rats are used asplacebo-treated controls. The remaining OVX animals are treated with 3to 5 doses of test drug for a period of 5 weeks (35 days). As a positivecontrol, a group of OVX rats can be treated with an agent such as PTH, aknown anabolic agent in this model (Kimmel, et al., Endocrinology (1993)132:1577-84). To determine effects on bone formation, the followingprocedure can be followed. The femurs, tibiae and lumbar vertebrae 1 to4 are excised and collected. The proximal left and right tibiae are usedfor PQCT measurements, cancellous bone mineral density (BMD)(gravimetric determination), and histology, while the midshaft of eachtibiae is subjected to cortical BMD or histology. The femurs areprepared for pQCT scanning of the midshaft prior to biomechanicaltesting. With respect to lumbar vertebrae (LV), LV2 are processed forBMD (PQCT may also be performed); LV3 are prepared for undercalcifiedbone histology; and LV4 are processed for mechanical testing.

[0096] Assays for Hair Growth

[0097] The ability of the compositions of the invention to stimulatehair growth was, surprisingly, discovered in the course of assessingtheir ability to stimulate the growth of bone. Accordingly, set forthbelow is the bone growth assay that led to the discovery of the hairgrowth stimulating ability of these compounds.

[0098] In Vivo Assay of Effects of Compounds on Hair FolliclesProliferation and Hair Growth

[0099] The assay described above to assess the effect of compounds oncalvarial bone growth can also be used to assess the ability ofcompounds to stimulate hair growth. The test compound or appropriatecontrol vehicle is applied to the upper and lower back of male ICR Swisswhite mice either topically or by subcutaneous injection. The vehicle isselected as appropriate for the compound to be tested and for the routeof administration. Optionally, the hair in the test area may be removedprior to administration. After a suitable interval, typically 7 days,the mice are anesthetized and a biopsy of the dorsal treatment area istaken using a 6 mm dermal punch. The specimens are fixed in 10% bufferedformalin and imbedded in paraffin wax, and sectioned and stained toobserve hair follicles. In addition, photography can be used to observeand record hair growth; typically such growth is observed after 14-18days. After a suitable interval, typically 21 days, the animals may beeuthanized and the hair analyzed for fiber analysis and the tissue fromthe treatment area analyzed for quantitation of hair follicles.

[0100] In more detail, male ICR Swiss white mice, aged 4-6 weeks andweighing 13-26 gm, are employed, using 4-5 mice per group. The calvarialbone growth assay is performed as described above. Briefly, the testcompound or appropriate control vehicle is injected into thesubcutaneous tissue over the right calvaria of normal mice. Typically,the control vehicle is the vehicle in which the compound wassolubilized, and is PBS containing 5% DMSO or is PBS containing Tween (2μl/10 ml). The animals are sacrificed on day 14 and bone growth measuredby histomorphometry. Bone samples for quantitation are cleaned fromadjacent tissues and fixed in 10% buffered formalin for 24-48 hours,decalcified in 14% EDTA for 1-3 weeks, processed through gradedalcohols; and embedded in paraffin wax. Three to five μm sections of thecalvaria are prepared, and representative sections are selected forhistomorphometric assessment of the effects on bone formation and boneresorption. Sections are measured by using a camera lucida attachment totrace directly the microscopic image onto a digitizing plate. Bonechanges are measured on sections cut 200 μm apart, over 4 adjacent 1×1mm fields on both the injected and noninjected sides of the calvaria.New bone is identified by its characteristic woven structure, andosteoclasts and osteoblasts are identified by their distinctivemorphology. Histomorphometry software (OsteoMeasure, Osteometrix, Inc.,Atlanta) is used to process digitizer input to determine cell counts andmeasure areas or perimeters.

[0101] Typical treatment regimens for testing utilize application of thecompound to be tested over several days of repeated administration.

[0102] Nature of the Compounds Useful in the Invention

[0103] The compounds useful in the methods and compositions of theinvention are inhibitors of proteasomal activity, of the transcriptionfactor NF-κB, preferably both. Known inhibitors of these activities canbe ascertained from the literature or compounds can be tested for theseactivities using assays known in the art. In addition, inhibitors whichlower the level of effective expression of the nucleotide sequenceencoding the enzymes that have proteasomal activity or of the nucleotidesequence encoding NF-κB can be assessed and used in the inventionmethods.

[0104] The compounds thus identified, which are used according to themethod of the invention as it relates to treating bone defects, however,preferably do not include compounds that inhibit the isoprenoid pathway,such as the statins. A description of these excluded compounds can befound in WO98/25460 and in U.S. Ser. No. 09/096,631, both cited aboveand incorporated herein by reference. For convenience, the isoprenoidpathway referred to is set forth herein in FIG. 1. One class ofcompounds which are inhibitors are the statins which have the formula

[0105] wherein X in each of formulas (1) and (2) represents asubstituted or unsubstituted alkylene, alkenylene, or alkynylene linkerof 2-6C;

[0106] Y represents one or more carbocyclic or heterocyclic ringswherein, when Y comprises two or more rings, said rings may be fused;and

[0107] R′ represents a cation, H or a substituted or unsubstituted alkylgroup of 1-6C; and

[0108] the dotted lines represent optional π-bonds.

[0109] These compounds may, however, be used in the method of theinvention as it relates to the stimulation of hair growth.

[0110] Compounds known to be proteasome or NF-κB inhibitors include:Proteasome Inhibitors PSI (and its epoxide)N-carbobenzoyl-Ile-Glu-(OtBu)-Ala-Leu-CHO MG-132N-carbobenzoyl-Leu-Leu-Leu-CHO MG-115 N-carbobenzoyl-Leu-Leu-Nva-CHOMG-101 or Calpain Inh I N-Acetyl-Leu-Leu-norLeu-CHO ALLMN-Acetyl-Leu-Leu-Met-CHO N-carbobenzoyl-Gly-Pro-Phe-Leu-CHON-carbobenzoyl-Gly-Pro-Ala-Phe-CHO N-carbobenzoyl-Leu-Leu-Phe-CHON-carbobenzoyl-Leu-Ala-Leu-CHO Gliotoxin

SN50 NLS of NF-κB MW 2781 Bay 11-7082

Capsaicin

PDTC

ALLN

MG-262

PPM-18

Cyclosporin A

Epoxomicin

[0111] See, for example, Vinitsky, A., et al., J Biol Chem (1994)269:29860-29866; Figueiredo-Pereira, M. E., et al, J Neurochem (1994)63:1578-1581; Wojcik, C., et al., Eur J Cell Biol (1996) 71:311-318.

[0112] In the foregoing list, lactacystin is known to be an irreversibleinhibitor of proteasome activity. It binds to the β catalytic subunitand is a specific inhibitor of the 20S proteasome. It also irreversiblyinhibits NF-κB.

[0113] SN50 is the NLS (nuclear localization sequence) of p50 plus thehydrophobic region of K-FGF. It inhibits the translocation of the NF-κBactive complex to the nucleus.

[0114] Certain peptidyl epoxy ketones such as EST and the epoxide of PSIare irreversible inhibitors of the proteasomes.

[0115] A particularly useful epoxy ketone is epoxomicin, a naturalproduct whose structure is shown in the above table. It appears to be ahighly specific and irreversible inhibitor of the proteasome which hasbeen shown to modify, covalently, at least four catalytic subunits ofthe 20S proteasome. It does not appear to inhibit nonproteasomalproteases such as cathepsin B, papain, chymotrypsin or calpain atconcentrations up to 50 μM. Epoxomicin also effectively NF-κB activationin vitro. The synthesis of epoxomicin is described by Sin, N., et al.,Biorg Med Chem Lett (1999) 9:2283-2288.

[0116] MG-132 shows activity against the chymotryptic activity of the20S protein without affecting its ATPase or isopeptidase activity andreversibly inhibits NF-κB activity. MG-115 and MG-341 show similaractivities to MG-132. Various other inhibitors of NF-κB are less activein the ABA assay. These include capsaicin, curcumin, andresiniferatoxin. Other compounds known to inhibit NF-κB are gliotoxinand PDTC (1-pyrrolidine carbothiotic acid). Various other compounds suchas BAY-11-7082 and BAY-11-7085 as well as calyculin-A inhibitphosphorylation of NF-κB. Calpain inhibitor inhibits calpain 1 and theproteasome; other compounds such as olomoucine and roscovitine inhibitcdk2 and/or cdk5.

[0117] An additional compound shown to be a proteasome inhibitor ispentoxyfilline (PTX). Combaret, L., et al., Mol Biol Rep (1999)26:95-101. It is active in the in vitro calvarial assay described above.

[0118] As set forth above, in preferred embodiments of the methods ofthe invention, the identified compounds used in treatment of bonedisorders are other than statins and other compounds that inhibit theisoprenoid pathway, typically as shown in FIG. 1. In other preferredembodiments, also excluded from use in the methods of treatment of bonedisorders of the present invention, are compounds described in PCTapplications WO98/17267, WO97/15308, and WO97/48694 cited andincorporated herein by reference hereinabove. However, the use of thesecompounds in the method to stimulate hair growth according to theinvention is not excluded.

[0119] One particular type of compounds that can be used in the methodsand pharmaceutical compositions for treating pathological dentalconditions or degenerative joint conditions in a vertebrate animal arecompounds that inhibit the chymotrypsin-like activity of the proteasome.Any known chymotrypsin-like activity inhibitors can be used. Forexample, the compound used can have a “warhead,” i.e., a functionalgroup, that reacts with the chymotrypsin-like site of the proteasome.Exemplary “warheads” include an epoxide that is capable of forming amorpholino ring with the threonine residue of the chymotrypsin-likecatalytic site of the proteasome (Elofsson, et al., Chemistry & Biology,6:811-822 (1999); and Groll, et al., J Am. Chem. Soc., 122:1237-1238(2000)), or a group that can react irreversibly with the active site ofthe chymotrypsin-like activity such as a —B(OR)₂ group, a —S(OR)₂ group,or a —SOOR group, wherein R is H, an alkyl (C₁₋₆) or an aryl (C₁₋₆). Inone specific embodiment, the compound used is a peptide, or an analogthereof, having the above-described “warhead.” Preferably, the peptidehas at least 3 amino acids.

[0120] One example of the chymotrypsin-like activity inhibitors that canbe used is a peptide α′,β′-epoxyketone. The length of the peptide can be3, but is preferably at least 4 amino acids. The C-terminus amino acidof the peptide α′, β′-epoxyketone is preferably a hydrophobic amino acidsuch as leucine or phenylalanine. More preferably, the peptideα′,β′-epoxyketone used has the following formula:

[0121] wherein R, R¹, R² and R are independently, for example,

[0122] Preferably, the peptide α′,β′-epoxyketone has the followingstereo-configuration:

[0123] In the exemplary compounds having the above formula R² and R³ arethe side chains of leucine, isoleucine, valine or phenyl alanine andthese include

[0124] Another example of the peptide α′,β′-epoxyketone that can be usedhas the following formula:

[0125] wherein R can be, for example,

[0126] Preferably, the peptide α′,β′-epoxyketone has the followingstereo-configuration:

[0127] More preferably, the peptide α′,β′-epoxyketone used is:

[0128] Still another example of the proteasome inhibitors that can beused has the following formula:

[0129] wherein the warhead reacts irreversibly with the catalyticchymotrypsin site of the proteasome;

[0130] A is independently CO—NH or isostere thereof;

[0131] R is independently a hydrocarbyl;

[0132] X is a polar group; and

[0133] n=0-2.

[0134] Optionally, the R group can contain a substituted group such as ahalo group, —OR, —SR, —NR2, ═O, —COR, —OCOR, —NHCOR, —NO₂, —CN, CF₃.Also optionally, X can be a protected group.

[0135] Alternatively, the following compounds can also be used:

[0136] epoxomicin, PS-341, NLVS, lactacystin, PTX, or a peptidylaldehyde. Particularly preferred is the epoxide of PSI.

[0137] In addition, proteasome inhibitors disclosed in U.S. Pat. No.5,780,454, which is incorporated by reference in its entirety herein,can be used. Especially, the proteasome inhibitors having formula (1b)or (2b), as disclosed in U.S. Pat. No. 5,780,454, can be used.

[0138] Compounds that inhibit the trypsin-like or PGPH activity of theproteasome can be used in the methods and pharmaceutical compositionsfor stimulating hair growth in a mammalian subject. Preferably, thecompound used is lactacystin or a peptidyl aldehyde.

[0139] Other compounds that can be used in the present methods andpharmaceutical compositions for treating pathological dental conditionsor degenerative joint conditions in a vertebrate animal or forstimulating hair growth in a mammalian subject include, but are notlimited to, sulfasalazine (Liptay, et al., Br. J Pharmacol.,128(7):1361-9 (1999)); and Wahl, et al., J Clin. Invest., 101(5):1163-74(1998)) and calpain inhibitor II.

[0140] The following examples are intended to illustrate but not tolimit the invention.

EXAMPLE 1 High Throughput Screening

[0141] Thousands of compounds have been tested in the assay system setforth in U.S. Ser. No. 08/458,434, filed Jun. 2, 1995, and incorporatedherein by reference. Representative compounds of the invention gavepositive responses, while the majority of (unrelated) compounds areinactive. In this screen, the standard positive control was the compound59-0008 (also denoted “OS8”), which is of the formula:

[0142] In more detail, the 2T3-BMP-2-LUC cells, a stably transformedosteoblast cell line described in Ghosh-Choudhury et al. Endocrinology(1996) 137:331-39, referenced above, was employed. The cells werecultured using α-MEM, 10% FCS with 1% penicillin/streptomycin and 1%glutamine (“plating medium”), and were split 1:5 once per week. For theassay, the cells were resuspended in a plating medium containing 4% FCS,plated in microtiter plates at a concentration of 5×10³ cells (in 50μl)/well, and incubated for 24 hours at 37° C. in 5% CO₂. To initiatethe assay, 50 μl of the test compound or the control in DMSO was addedat 2× concentration to each well, so that the final volume was 100 μl.The final serum concentration was 2% FCS, and the final DMSOconcentration was 1%. Compound 59-0008 (10 μM) was used as a positivecontrol.

[0143] The treated cells were incubated for 24 hours at 37° C. and 5%CO₂. The medium was then removed, and the cells were rinsed three timeswith PBS. After removal of excess PBS, 25 μl of 1× cell culture lysingreagent (Promega #E153A) was added to each well and incubated for atleast ten minutes. Optionally, the plates/samples could be frozen atthis point. To each well was added 50 μl of luciferase substrate(Promega #E152A; 10 ml Promega luciferase assay buffer per 7 mg Promegaluciferase assay substrate). Luminescence was measured on an automated96-well luminometer, and was expressed as either picograms of luciferaseactivity per well or as picograms of luciferase activity per microgramof protein.

[0144] In this assay, compound 59-0008(3-phenylazo-1H-4,1,2-benzothiadiazine) exhibits a pattern of reactivitywhich is maximal at a concentration of approximately 3-10 μM.Accordingly, other tested compounds can be evaluated at variousconcentrations, and the results compared to the results obtained for59-0008 at 10 μM (which value would be normalized to 100).Alternatively, the reactivity of a compound to be tested can be compareddirectly to a negative control containing no compound.

[0145] The control compound 59-0328, which is simvastatin, gives a goodresponse. The known proteasome inhibitors MG-132 and MG-115 also showhigh activity; MG-132 is effective at lower concentrations. Positiveresponses are also obtained using lactacystin. However, gliotoxin,olomoucine, roscovitine, SN50, PDTC, and capsaicin do not give promisingresponses.

EXAMPLE 2 In vitro Bone Formation

[0146] Selected compounds and appropriate controls were assayed in vitro(ex vivo) for bone formation activity (described above in “Techniquesfor Neonatal Mouse Calvaria Assay (in vitro)). Histomorphometricalassessments of ex vivo calvaria were carried out using an OsteoMetricsbone morphometry measurement program, according to the manufacturer'sinstructions. Measurements were determined using either a 10- or 20-foldobjective with a standard point counting eyepiece graticule.

[0147] New bone formation was determined (using a 10× objective) bymeasuring the new bone area formed in one field in 3 representativesections of each bone (4 bones per group). Each measurement was carriedout {fraction (1/2)} field distance from the end of the suture. Bothtotal bone and old bone area were measured. Data were expressed as newbone area in μm².

[0148] The results in Example 1 were somewhat imperfectly correlatedwith the results in this assay. The control compound, simvastatin showednew bone formation in this assay as did MG-132 and lactacystin. MG-115also showed positive results although less dramatic than those ofsimvastatin. However, gliotoxin, which appeared negative in the ABAassay of Example 1 did demonstrate the ability to stimulate bone growth.The remaining compounds, olomoucine, roscovitine, SN50, PDTC andcapsaicin appeared negative in this assay.

[0149] Osteoblast numbers are determined by point counting. The numberof osteoblast cells lining the bone surface on both sides of the boneare counted in one field using a 20× objective. Data are expressed asosteoblast numbers/mm of bone surface.

[0150] Alkaline phosphatase activity is measured in the conditionedmedia of the murine organ cultures, using the method described byMajeska, R. J., et al., Exp Cell Res (1978) 111:465-465. Conditionedmedia are incubated at 37° C. for 20 minutes with phosphatase substrate104 (Sigma) and the reaction stopped with 2 ml of 0.1 M NaOH. Alkalinephosphatase activity is calculated by measuring cleaved substrate at anoptical density of 410 nm in a Beckman dual beam spectrophotometer fromthe OD410 and corrected for protein concentration.

[0151] PSI and MG-132 and control compounds/factors bFGF and BMP-2, anda vehicle control were tested in this assay and the calvaria wereanalyzed histomorphometrically, as described above. Increase in bonearea as a function of concentration; the increase in osteoblasts and theenhancement of alkaline phosphatase activity for PSI were measured.

[0152] The data show that PSI is as good as, or better than, BMP-2 andbFGF (two “gold standard” agents for bone growth; see Wozney, J., MolecReprod Dev (1992) 32:160-67; W095/24211) for inducing bone formation.

[0153] An additional experiment, pentoxyfilline (PTX) was tested in theforegoing assay. It exhibited the ability to enhance new bone formationin concentrations as low as 0.1 μm. At a concentration of 10 μm, PTXappeared to enhance the new bone are over control by over 100%; at 100μm, the increase was approximately three (3) times that of control.

EXAMPLE 3 In vivo Calvarial Bone Growth Data

[0154] PSI and MG-132 were assayed in vivo according to the proceduredescribed previously (see “In vivo Assay of Effects of Compounds onMurine Calvarial Bone Growth”, supra). As a control, simvastatinprovided a 1.5 fold increase in the number of osteoblasts.

[0155] In one experiment, vehicle control, bFGF and varying doses of PSIwere tested in the in vivo calvarial bone growth assay. The results arereported as a measurement of total bone area, % increase in area overvehicle control, and % increase in new bone width as shown below. %Increase* in Total Bone Bone Area Compared % Increase* in Compound Area(μm²) with Control New Bone Width Control 0.64 ± 0.03 0.1 mg/kg/day 0.74± 0.02 21.7 ± 3.5   1 mg/kg/day 0.83 ± 0.02 35.4 ± 3.4 19.9 ± 2.0   5mg/kg/day 0.79 ± 0.03 32.1 ± 5.6 19.9 ± 4.4 *p < 0.05 *p < 0.001

[0156] In addition, histological examination showed confirmation of bonegrowth both when 5 mg/kg/day of PSI was used and 1 mg/kg/day was used.

EXAMPLE 4 Summary of Effects on Bone Formation

[0157] The table below summarizes the results obtained for compoundstested in the various assays set forth above. It is seen that compoundsthat are proteasome inhibitors also enhance bone formation. In thecompounds tested in this table, however, compounds which are known to beinhibitors only of NF-κB but which fail to inhibit proteasomal activity,do not enhance luciferase activity (indicative of BMP-2 promoteractivity) in the high through-put assay, nor do they enhance boneformation in the calvarial assay in vitro, to as great an extent as doproteasome inhibitors. Compounds useful in the invention include:Compound Structure Simvastatin

Lactacystin

PS1 Z-lie-Glu(OtBu)-Ala- Leu-CHO MG-132

MG262

MG115

ALLN

Cyclosporin A

Gliotoxin

SN50 NLS of NF-KB MW 2781 N-Acetyl-Leu-Leu Meth-CHO PPM-18

Bay 11-7082

Capsaicin

PDTC

Luciferase Bone Proteasome Activity Formation Activity Compound(ED₅₀-μM) (ED₅₀-μM) (ED₅₀-μM) Simvastatin

0.2

0.2

— Lactacystin

1

1

1.5 PS1

0.05

0.03

0.035 MG-132

0.25

0.5

0.3 MG262

0.1

0.1

0.07 MG115

2

1

1 ALLN

10 — —

1.5 Cyclosporin A — —

10

1.0 Gliotoxin — —

10 — — SN50 — — — — — — — — — —

4 PPM-18 — — — — — — Bay 11-7082 — — — — — — Capsaicin — — — —

30 PDTC — — — — — —

EXAMPLE 5 Confirmation of Proteasomal Inhibition

[0158] An assay to test the effect of compounds on the 20S thermophilaproteasome activity was employed. Purified 20S thermophila proteasomesand the fluorogenic peptide substrate Suc-Leu-Leu-Val-Tyr-AMC areavailable from CalBiochem, San Diego, Calif. Briefly, serial dilutionsof the inhibitor to be tested were mixed with proteasome solution at aconcentration of proteasome of 0.01 mg/ml. After 30 min incubation at37° C., substrate solution at a final concentration of 25-30 μg/ml wasadded and the mixture incubated at 37° C. and then read at 15 min, 30min, and 60 min in a Fluoroscan instrument. The percentage diminution influorescence in the presence as compared to the absence of inhibitor isthen calculated.

[0159] Epoxomicin, PSI and MG-132 were tested in this assay with theresults shown below in Table 1 which sets forth the percent proteasomalactivity in the presence of various concentrations of these compounds ascompared to untreated control. TABLE 1 Epoxomicin PSI MG-132 Dose %Proteasomal % Proteasomal % Proteasomal (μM) Activity Activity Activity2    — — 19 1     5  5 44 0.5   6  6 57  .25  14 11 73 0.125 30 30 870.062 51 54 90 0.032 71 76 98 0.016 85 86 100  0.008 95 94 101  0.004102  95 99 0.002 103  100  101 

[0160] As seen, both epoxomicin and PSI were able to diminishproteasomal activity by approximately 50% at a concentration of about 60nM. In addition, proteasome inhibitor I (PSI) gave a dose response curvesimilar to that of epoxomicin.

EXAMPLE 6 Additional High Throughput Assay

[0161] The high through-put assay described in Example 1 was performedas an independent experiment to test the activity of epoxomicin, PSI andwith simvastatin as a standard positive control. The results of thisassay are shown in Table 2. TABLE 2 Epoxomicin PSI Simvastatin DoseLuciferase Luciferase Luciferase (μM) activity/well activity/wellactivity/well 10 — — 0.15 5 — — 0.15 2.5 — — 0.14 1.25 — — 0.11 0.625 —— 0.09 0.32 — — 0.07 0.16 — — 0.07 0.08 0.21 0.18 0.07 0.04 0.20 0.120.07 0.02 0.17 0.09 0.07 0.01 0.14 0.08 0.07 0.005 0.10 0.08 0.07 0.00250.08 0.07  0.065 0.00125 0.07 0.08 0.07 0.000625 0.07 0.07 0.07

[0162] Both epoxomicin and PSI were more active than simvastatin in thisassay.

EXAMPLE 7 Activity of Epoxomicin in the Calvarial Assay

[0163] The calvarial assay described in Example 3 was performed to testthe ability of epoxomicin to stimulate bone growth. The results in termsof new bone area are shown in Table 3. As seen, at a concentrations of5-10 nM, significantly more bone was formed than in the control. TABLE 3Dose New Bone Area (μM) (mm² × 10⁻³) S.E.M. Control 2.76 0.40 0.01  5.74 # 0.27 0.005   6.54 # 0.45 0.0025 4.04 0.39 0.00125 2.80 0.840.000625 2.78 0.50

EXAMPLE 8 Effect of PSI and Other Proteasome Inhibitors on Hair FollicleProduction

[0164] The in vivo bone calvarial growth assay of Example 3 was modifiedto observe the number of hair follicles in treated mice. In initialobservations, PSI (5 mg/kg/day) was injected three times a day for 5days over the calvaria of Swiss ICR mice as described above. Sixteendays later the mice were sacrificed. Histology of the calvaria revealeda strikingly large increase in the number of hair follicles in thosemice treated with PSI versus control mice. In addition to PSI, MG-132(10 mg/kg), MG115 (10 mg/kg) and lactacystin administered in the sameway also stimulated an increase in the number of hair follicles.

EXAMPLE 9 Stimulation of Hair Growth

[0165] Male Swiss ICR mice were first treated to remove hair from thescalp and dorsal regions as follows. Paraffin wax was liquefied byheating to 55° C. and the liquefied wax then applied by brush to thescalp and/or back (under anesthesia). The wax was allowed to solidifyand then removed. The day following hair stripping, PSI (1 mg/kg/day)was injected subcutaneously three times a day for five days into thescalp and dorsal region. On day 7 a dermal punch biopsy was taken;histology revealed a large increase in the number of hair follicles inmice administered PSI versus control mice. By day 18, it was observablethat the treated mice had a hair growth rate greater than that of themice in the control group.

[0166] The mice were sacrificed on day 21 and histology was performed onthe dermis of the scalp and of the dorsal region. In the treated mice,mature hair follicles in numbers much greater than in controls hadmigrated to the lower region of the dermis. Upon closer examination, itwas observed that mice that had received only vehicle had quiescent hairfollicles. When treated with PSI such follicles were stimulated todifferentiate into mature hair follicles and to migrate to the lowerregion of the dermis.

EXAMPLE 10 Effects on Subcutaneous Tissue

[0167] Effects of PSI on subcutaneous tissue of the scalp of 6-weeks oldICR mice from longitudinal and transverse sections were determined. PSIdissolved in 50% propylene glycol, 10% DMSO, and 40% distilled water wasinjected daily for 5 days (1 mg/kg body weight/day) into thesubcutaneous tissue, and the tissue examined histologically 16 dayslater. The numbers of hair follicles increased and the downwardextension of these hair follicles into the dermal tissue (100×) wasnoted, which are both hallmarks of anagen. There was an obvious increasein size of the follicle diameter and the root sheath diameter (200×).

EXAMPLE 11 Explants

[0168] Cultured skin explants from 5 day old mice cultured for 72 hourswere treated with proteasome inhibitors, non-proteasomal proteases andnon-proteasomal inhibitors of NF-κB and effects on hair folliclediameter and follicle elongation were determined according to the methodof Kamiya, T., et al., J Derm Sci (1998) 17:54-60. Skin slices from thedorsal skin of 5 day old C3H/HeSlc mice were cultured in I ml of αMEMand 0.1% BSA for 72 hours and then assessed for changes in hair folliclediameter and hair elongation under an inverted microscope, using imageanalysis. The following doses were used—epoxomicin (2.5 nM), PS1-epoxide(12.5 nM), PS1 (12.5 nM), MG-132 (0.5 μM), PDTC (10 μM), and Roscovotine(10 μM). While the NF-κB inhibitor 1-pyrrolidinecarbodithioic acid(PDTC) and the cyclin-dependent kinase inhibitor roscovitine did notsignificantly affect follicle growth, the proteasome inhibitorslactacystin, PS1, and MG132 effectively stimulated hair follicledifferentiation and enhanced hair growth. The epoxyketone-containingnatural product epoxomicin, which specifically inhibits thechymotrypsin-like catalytic activity of the proteasome was found to beeffective in concentrations as low as 12.5 nM. A hybrid compound thatcontained the peptide side-chain of PSI linked to the epoxyketonepharmacophore of epoxomicin to give it selectivity for the proteasome(PS 1-epoxide) selectively inhibits the chymotrypsin-like activity ofthe proteasome and potently stimulated new hair growth. Thus, thechymotrypsin-like activity of the proteasome is the catalytic componentof the proteasome that is responsible for the effects of these compoundson hair follicle differentiation and hair growth. Only proteasomeinhibitors had discemable effects on these parameters.

EXAMPLE 12 Anagen Effect

[0169] Inhibitors of the proteasomal chymotrypsin-like activity weretreated for their capacity to induce transit of hair follicles intoanagen in vivo in C57 black mice eight weeks of age. Epoxomicin, PS 1 (5mg/kg/day) or PS1-epoxide (10 mg/kg/day) was injected subcutaneously inthe scalp daily for 5 days, and the underlying tissue examined 16 dayslater. All three compounds increased hair follicle differentiation invivo. In contrast, NF-κB inhibitors calpain inhibitor-I PDTC and2-benzoylamino-1,4-naphthoguinone (PPM-18), as well as the statinslovastatin and simvastatin, have no effects on proteasomal activity(Law, R. E, et al., Mol Cell Biol (1992) 12:103-111; Guijarro, C., etal, Nephrol Dial Transplant (1996) 11:990-996). Lovastatin andsimvastatin injected locally into the subcutaneous tissue in sufficientconcentrations to cause local periosteal bone formation (Mundy, G., etal., Science (1999) 286:1946-1949), had no effect on hair follicles.Calpain inhibitor-I, PDTC and PPM-18 also had no effect on hairfollicles.

[0170] These data suggest that only those compounds that causeinhibition of proteasome function stimulate induction of anagen inquiescent hair follicles in vivo.

EXAMPLE 13 Topical Administration

[0171] PSI was prepared as a topical formulation, where the vehicle was50% propylene glycol, 30% ethanol, 20% deionized water, at 0.1%concentration of PSI. The solution was applied 3 times a day for 5 days.The mice in a treated group were observed as compared to controlssimilarly treated with vehicle alone. The results at day 16 showedstimulation of hair growth relative to the controls.

[0172] In addition to stimulating hair growth, PSI was able to thickenboth the hair and the hair shaft. PSI increases hair count when thefollicle area is greater than 0.01 mm². When the protocol above wasrepeated using a 0.5% solution of PSI in groups containing 5 mice each,the number of hairs per 0.8 mm² was 60 in the treated mice versus about10 in the control group. The percentage of follicle area in a region ofabout 0.8 mm² was about 30% as an average in the treated group ascompared to 15% as an average in the control group.

EXAMPLE 14 Dose Requirements

[0173] In order to determine the minimal effective dosage of PSI, whenused topically, a dose response curve for PSI was prepared. Allexperiments were preformed according to current good laboratory practiceregulations (21 CFR58). The mice were divided into 7 groups, 10 miceeach, wherein one group was control treated only with vehicle and groups1-6 with a series of increasing concentrations of PSI in a vehiclecomprising 50% propylene glycol, 30% ethanol, 20% deionized water. Theconcentrations were 0.006%, 0.012%, 0.025%, 0.05%, 0.11% and 0.5%.

[0174] The mice were anesthetized (50 μl Mouse Cocktail containing 3 mlketamine, 2 ml small animal rompum, 5 ml NaCl), identified by ear punchcode, weighed and the hair on the dorsal side removed by waxing asdescribed in Example 6. After waxing, the animals were photographed. Onthe following day (day 1), 100 μl of PSI at the above concentrations invehicle was brushed onto the area of removed hair. A similar applicationof PSI solution was performed daily for an additional 4 days.

[0175] On Day 7 mice were anesthetized and a biopsy of the dorsaltreatment area taken using a 6 mm dermal punch; the specimens were fixedin 10% buffered formalin and embedded in paraffin wax. Sections were cutusing a standard microtome.

[0176] Mice were monitored daily for signs of hair growth, and any hairgrowth was recorded by photography. On day 21 animals were euthanized(75 mg/kg body weight phenobarbital, IP injection), 2 cm hair sampleswere taken for optical based fiber analysis, and the remaining dorsaltreatment area was fixed in 10% buffered formalin for furtherhistological analysis. Analysis included quantification of hairthickness and quantification of mature hair follicles. Results wereexpressed as the mean=+/− the standard error of the mean. Data wereanalyzed by repeated measures of analysis of variance followed by theTukey-Kramer post test P values of <0.05 were considered significant.

[0177] The results indicate that the minimal effective dose of PSI is0.5% applied 1 time a day for 4 days; additional experiments showed that0.1% of PSI applied topically 3 times a day for 5 days was alsoeffective.

[0178] Gross observation of mice receiving an effective dose indicatedan enhanced rate of hair growth, a thickening of hair diameter, increasein sheath diameter, and differentiation of quiescent hair follicles intomore mature forms.

EXAMPLE 15 Effect Without Depilation

[0179] To confirm that the responses described above occurred in theabsence of depilation and in other strains of mice, PSI (0.5%) wasapplied topically once daily for 5 days to eight week old male C57 BL/6mice in which the dorsal trunks were shaved but not depilated. For thisexperiment, the hair of 18 C57 BL/6 mice was carefully clipped the hairfrom the dorsal trunks. Three groups were tested. One group was treatedwith PSI (0.5%) applied topically to the shaved area daily. In thesecond group, PS1 (1 mg/kg body weight/day) was injected locally intothe subcutaneous tissue of the shaved area. These two groups werecompared with the third group, which was untreated other than the hairon the dorsal trunk was similarly clipped. Blonde hair dye (extrastrength bleach powder and 12% hydrogen peroxide in a 1 to 1 w/v ratio)was applied to the dorsal trunks of all mice on the seventh day in orderto make it easier to detect new hair growth represented by appearance inthe blonded areas of the natural black hair of the mice associated withinduction of melanogenesis. By the sixteenth day, the differencesbetween the treated and the untreated mice were obvious, with black hairappearing on the dorsal trunks of the treated mice receiving eithertopically applied or injected PSI, indicating the more rapid growth ofnew hair at the site of drug application in these mice. Transversesections taken from the dorsal trunks of male C57 BL/6 mice directlyfrom areas of application as well as areas adjacent to and distant fromthat site showed stimulation of hair sheaths was limited to areas ofdirect topical application. Transverse sections of skin from the dorsaltrunks of these mice showed that PSI increased both the diameter of thehair and the diameter of the inner and outer root sheath only in theapplied areas.

EXAMPLE 16 Effect of Hair Cycle Stage

[0180] Skin explants from mice during telogen at 3 weeks of age werecultured for 72 hours, in the presence of proteasome inhibitors, andthen the explants were histologically examined. In the untreated controlexplants, the follicles were few and small. In the explants treated withPS1 (50 nM), epoxomicin (25 nM), and the hybrid PS1-epoxide (50 nM),there was an obvious increase in hair follicle diameter, and follicleelongation. Minoxidil also caused these changes, but at concentrations10,000-fold greater. Since these small follicles resemble theminiaturized follicles seen in male pattern baldness, this suggeststhese compounds may have the same effects in that situation.

[0181] The contents of all documents cited above are expresslyincorporated herein to the extent required to understand the invention.

[0182] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1. A method to enhance bone formation or to treat pathological dentalconditions or to treat degenerative joint conditions in a vertebrateanimal, which method comprises administering to a vertebrate subject inneed of such treatment an effective amount of a compound that inhibitsthe activity of NF-κB or that inhibits proteasomal activity or thatinhibits production of proteasome proteins wherein the compound does notinhibit the isoprenoid pathway.
 2. The method of claim 1, wherein thecompound inhibits proteasomal activity or inhibits production ofproteasomal proteins.
 3. The method of claim 2, wherein the compoundinhibits the chymotrypsin-like activity of the proteasome.
 4. The methodof claim 3, wherein the compound is a peptide having at least 3 aminoacids and a C-terminal functional group that reacts with the threonineresidue of the chymotrypsin-like catalytic site of the proteasome. 5.The method of claim 4, wherein the c-terminal functional group isselected from the group consisting of an epoxide, a —B(OR)₂ group, a—S(OR)₂ group and a —SOOR group, wherein R is H, an alkyl (C₁₋₆) or anaryl (C₁₋₆).
 6. The method of claim 5, wherein the functional group isan epoxide that forms a morpholino ring with the threonine residue ofthe chymotrypsin-like catalytic site of the proteasome.
 7. The method ofclaim 3, wherein the peptide is a peptide α′,β′-epoxyketone.
 8. Themethod of claim 7, wherein the peptide α′,β′-epoxyketone has at least 4amino acids.
 9. The method of claim 7, wherein the c-terminus amino acidof the peptide α′,β′-epoxyketone is a hydrophobic amino acid.
 10. Themethod of claim 9, wherein the hydrophobic amino acid is leucine orphenylalanine.
 11. The method of claim 7, wherein the peptideα′,β′-epoxyketone has the following formula:

wherein each of R, R¹, R² and R³ is a hydrophophic substituent.
 12. Themethod of claim 11 wherein each of R, R¹, R² and R³ is independentlyselected from the group consisting of


13. The method of claim 11, wherein R² and R³ are

and the compound is selected from the group consisting of


14. The method of claim 11, wherein the peptide α′,β′-epoxyketone hasthe following stereo-configuration:


15. The method of claim 7, wherein the peptide α′,α′-epoxyketone has thefollowing formula:

wherein R is selected from the group consisting of


16. The method of claim 15, wherein the peptide α′,β′-epoxyketone hasthe following stereo-configuration:


17. The method of claim 16, wherein the peptide α′,β′-epoxyketone is


18. The method of claim 3, wherein the compound is selected from thegroup consisting of

, epoxomicin, PS-341, NLVS, PSI epoxide, lactacystin, PTX and a peptidylaldehyde.
 19. The method of claim 3, wherein the compound has thefollowing formula:

wherein the warhead reacts irreversibly with the catalytic chymotrypsinsite of the proteasome; A is independently CO—NH or isostereomerthereof, R is independently a hydrocarbyl; X is a polar group; andn=0-2.
 20. The method of claim 19, wherein R contains a substitutedgroup selected from the group consisting of a halo group, —OR, —SR,—NR₂, ═O, —COR, —OCOR, —NHCOR, —NO₂, —CN, and —CF₃.
 21. The method ofclaim 19, wherein X is protected.
 22. The method of claim 1, wherein thesubject is characterized by a condition selected from the groupconsisting of osteoporosis, bone fracture or deficiency, primary orsecondary hyperparathyroidism, periodontal disease or defect, metastaticbone disease, osteolytic bone disease, post-plastic surgery,post-prosthetic joint surgery, and post-dental implantation.
 23. Themethod of claim 1, which further comprises administering to the subjectone or more agents that promote bone growth or that inhibit boneresorption.
 24. The method of claim 23, wherein the agents are selectedfrom the group consisting of bone morphogenetic factors, anti-resorptiveagents, osteogenic factors, cartilage-derived morphogenetic proteins,growth hormones, estrogens, bisphosphonates, statins and differentiatingfactors.
 25. A method to treat a mammalian subject for a conditionbenefited by stimulating hair growth which method comprisesadministering to said mammalian subject in need of such treatment aneffective amount of a compound that inhibits the activity of NF-κB orthat inhibits proteasomal activity or that inhibits production of theseproteins.
 26. The method of claim 25, wherein said compound inhibitsproteasomal activity or inhibits production of proteasome proteins. 27.The method of claim 26, wherein the compound inhibits the trypsin-likeor PGPH activity of the proteasome.
 28. The method of claim 25, whereinthe compound is lactacystin or a peptidyl aldehyde.
 29. A pharmaceuticalcomposition for treating bone disorders, dental pathological conditionsor degenerative joint conditions, which composition comprises a compoundthat inhibits the activity of NF-κB or that inhibits proteasomalactivity or that inhibits production of these proteins, in the compounddoes not inhibit the isoprenoid pathway.
 30. The pharmaceuticalcomposition of claim 29, wherein the compound inhibits proteasomalactivity or inhibits production of proteasomal proteins.
 31. Thepharmaceutical composition of claim 30, wherein the compound inhibitsthe chymotrypsin-like activity of the proteasome.
 32. The pharmaceuticalcomposition of claim 31, wherein the compound is a peptide having atleast 3 amino acids and a c-terminal functional group that reacts withthe threonine residue of the chymotrypsin-like catalytic site of theproteasome.
 33. The pharmaceutical composition of claim 32, wherein thec-terminal functional group is selected from the group consisting of anepoxide, a —B(OR)₂ group, a —S(OR)₂ group and a —SOOR group, wherein Ris H, an alkyl (C₁₋₆) or an aryl (C₁₋₆).
 34. The pharmaceuticalcomposition of claim 32, wherein the peptide is a peptideα′,β′-epoxyketone.
 35. The pharmaceutical composition of claim 34,wherein the peptide α′,β′-epoxyketone has the following formula:

wherein each of R, R¹, R² and R³ is a hydrophophic substituent.
 36. Thepharmaceutical composition of claim 34, wherein the peptideα′,β′-epoxyketone has the following formula:

wherein R is selected from the group consisting of


37. The pharmaceutical composition of claim 31, wherein the compound hasthe following formula:

wherein the header reacts irreversibly with the catalytic chymotrypsinsite of the proteasome; A is independently CO—NH or isostereomerthereof; R is independently a hydrocarbyl; X is a polar group; andn=0-2.
 38. The pharmaceutical composition of claim 29, wherein thecompound is lactacystin, a peptidyl aldehyde, PTX, epoxomicin or PSIeopxide.
 39. A pharmaceutical composition for treating a conditionbenefited by stimulating hair growth, which composition comprises acompound that inhibits the activity of NF-κB or that inhibitsproteasomal activity or that inhibits production of these proteins. 40.The pharmaceutical composition of claim 39, wherein the compoundinhibits proteasomal activity or inhibits production of proteasomalproteins.
 41. The pharmaceutical composition of claim 40, wherein thecompound inhibits the trypsin-like or PGPH activity of the proteasome.42. The pharmaceutical composition of claim 39, wherein the compound islactacystin or a peptidyl aldehyde.
 43. A method to identify a compoundwhich enhances bone growth or stimulates hair growth, which methodcomprises subjecting said compound to an assay for determining itsability to inhibit NF-κB activity, whereby a compound which inhibits theactivity of NF-κB is identified as a compound which enhances bone growthor stimulates hair growth; or subjecting said compound to an assay fordetermining its ability to inhibit the production of NF-κB, whereby acompound which inhibits the production of NF-κB is identified as acompound which enhances bone growth or stimulates hair growth; orsubjecting a candidate compound to an assay to assess its ability toinhibit proteasomal activity, whereby a compound which inhibitsproteasomal activity is identified as a compound that enhances bonegrowth or stimulates hair growth; or subjecting a candidate compound toan assay to assess its ability to inhibit the production of enzymes withproteasomal activity, whereby a compound which inhibits the productionof enzymes with proteasomal activity is identified as a compound thatenhances bone growth or stimulates hair growth.
 44. The method of claim43, wherein the proteasomal activity to be inhibited is selected fromthe group consisting of the chymotrypsin-like activity, the trypsin-likeactivity, the PGPH activity and a combination thereof.